The resolution limit in traditional optical microscopy is limited by the numerical aperture of the microscope (NA) and the wavelength λ of imaging light, generally referred to as Raleigh limit or diffraction limit; and is equal to 0.61(NA)λ. This limit has recently been overcome in the case of fluorescence microscopy with Stimulated Emission Depletion (STED) microscopy by applying the statistical localization techniques of Photo Activation Localization Microscopy (PALM) and STochastic Optical Reconstruction Microscopy (STORM). These techniques are based on the physics of incoherently driven optical fluorescent transitions in dyes or other fluorescent molecules. In these techniques, light of one color turns off a fluorescent molecule, while light of another color is used to photo-stimulate the release of fluorescent photons producing an image of the molecules.
In STED microscopy, a first focused circular pulsed laser beam is used to provide an excited electronic state in fluorescent molecules at the focus of a microscope objective. Then a second pulsed laser beam of a different wavelength, focused to an annular shape is used to cause stimulated emission from the excited molecules in the annular spot de-excite the molecules back to the ground state manifold of the molecule. The annular beam is of the same size as the first beam but with a zero in the electric field at the center of the annulus of the stimulating beam. Unfortunately, this technique depends on the presence of efficient fluorescent molecules, where the fluorescence is often provided by a label rather than the intrinsic molecules. However, this technique is unable to identify intrinsic molecules that are not strongly fluorescent.
PALM and STORM work by turning off and on fluorescent emission from isolated dye molecules and finding the center position of the peak of emission of individual fluorescent molecules. These two techniques work best with a low concentration of fluorescent molecules.
Since not all molecules are strongly fluorescent, Raman microscopy is used to measure the vibration levels of intrinsic molecules in biological tissue, solid phase materials or on surfaces. In a Raman scattering process, a laser photon of a defined and stable wavelength is scattered from a molecule and shifted in wavelength by the vibrational energy level of a particular molecular bond. Raman spectroscopy and Raman microscopy typically operates with incident (excitation) light in the ultraviolet, visible or near infrared spectral regions which are weakly absorbed in many solvents such as water.
Conventional Raman microscopy has several drawbacks that have limited its application in biological imaging and hyper-resolution imaging, with hyper-resolution imaging defined as imaging with a resolution exceeding the diffraction limit. These include the following: 1) The incident laser light can stimulate fluorescent emission in the molecules under study, the solvent, or tissue under study which can coincide with the Stokes shifted Raman spectrum. 2) In general, the Raman process is inefficient. The collection efficiency of Raman scattered photons may be approximately 10−12. Since high intensity radiation of samples is limited by laser heating, Raman imaging and spectroscopy is a very slow process. 3) The scattering is non-directional, requiring very short working distances or very high numerical aperture lenses and microscope objectives. 4) Spectra of complex organic molecules may overlap, making it difficult to discriminate between different types of molecules. 5) Resolution is limited to that of the microscope which in general may be 0.4 microns or larger. 6) In small focal spots there are fewer molecules to produce the weak Raman scattering signal. 7) Poor scattering efficiency coupled with poor discrimination of signal and background makes detection of low concentrations of molecules impossible. 8) Raman transitions have very short excited state lifetimes. Virtual states for non-resonant Raman have femtosecond lifetimes, while Resonant Raman transitions have lifetimes in the 100 femtosecond range. These fast excited state decays make it impossible to use STED imaging techniques to achieve hyper-resolution imaging.
Variations of Raman imaging exist that can overcome several, but not all, of these deficiencies. For example, a longer excitation wavelength can be used to reduce background fluorescence; however, the molecular scattering cross section decreases with the inverse fourth power of the incident wavelength and the resolution in the image decreases with longer wavelength imaging. Alternatively, using resonant Raman spectroscopy can increase the Raman scattering cross section to 10−4 efficiency, whoever at the expense of significant enhanced fluorescent emission, and in tissues this is limited by the very strong background absorption in the ultraviolet (UV). The use of short laser pulses and time-gating the spectral acquisition to the sub-nanosecond regime may alleviate the adverse effects caused by fluorescence emission. However, most molecules have resonant absorption in the UV region which limits the use in plastic containers or the ability to see below the surface of biological tissues. Furthermore UV light often causes significant damage to biological and plastic materials, limiting its use to thin or surface samples.
A Raman technique that overcomes many of the aforementioned deficiencies is referred to as Coherent Anti-Stokes Raman Spectroscopy (CARS). CARS is a four-wave mixing process involving the generation of coherent vibration in the probed medium. Disadvantageously, the traditional CARS process produces a non-resonant incoherent background which can mask the measurement signal. This background is often caused by transitions involving solvent virtual levels. In addition, because the laser photons have to be tuned to the molecular transitions of interest, two or more different tunable picosecond lasers may be required. CARS also lacks adequate sensitivity, has a resolution of only about 300-400 nm, and cannot distinguish overlapping Raman bands.
Recently, Stimulated Raman Scattering (SRS) microscopy has demonstrated enhanced sensitivity over classical Raman microscopy, and similar Stimulated emission techniques have been shown to enhance the sensitivity of imaging with poorly fluorescent materials. These techniques rely on Stimulated emission of a traveling wave field to enhance the emission of light into the forward propagating laser field. This is the same process that produces “gain” in a laser beam propagating in a laser amplifier. However the femtosecond to picosecond lifetime of the excited states of these molecules have limited the application of STED techniques for use with these techniques for hyper resolution imaging.
When imaging biological cells and semiconductor devices, many of the structures of interest, such as chromosomes, ribosomes, membranes and transistor gates with dimensions below 100 nm, cannot be resolved with stimulated Raman and CARS microscopes.
It would therefore be desirable to provide an optical technique having an imaging resolution of better than 40 nm, for example, about 20-40 nm, with a high sensitivity and concurrent spectroscopic analysis. It would also be desirable to be able to image deeply below the surface which is not possible with resonance Raman techniques operating in the UV.